The present invention is in the field of nucleic acid detection methods, particularly the detection of RNA:DNA hybrids; and also in the field of proteins having RNA:DNA hybrid-binding activity.
Current detection methods of RNA:DNA hybrids include immunodetection methods using monoclonal antibodies. In one such immunodetection method, monoclonal antibodies are raised to a RNA:DNA heteropolymer duplex prepared by transcription of a single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity is selected. The antibody and an alkaline phosphatase-labeled second antibody are used to measure (via calorimetric response) hybrids formed between immobilized DNA probes of varying lengths and 23S ribosomal DNA. See, Boguslawski et al., “Characterization of Monoclonal Antibody to DNA:RNA and Its Application to Immunodetection of Hybrids,” J. Immunol Methods, 1986 May 1; 89(1): 123-30.
Several proteins bind to RNA:DNA hybrids, notably including RNA polymerases, which typically polymerize RNA from DNA templates, but also include reverse transcriptases, which typically polymerize DNA based upon RNA templates. Ribonuclease H(RNase H) biochemical activity also includes the binding of RNA:DNA hybrids. Several reverse transcriptases and polymerases have RNase H biochemical activity, including the exonuclease aspect of the activity.
RNases H are a ubiquitous enzyme family that is divided into two distinct phylogenetic sub-types, Type 1 and Type 2. The RNases H are unified by the common ability to bind a single-stranded (ss) RNA that is hybridized to a complementary DNA single strand, and then degrade the RNA portion of the RNA:DNA hybrid. While the RNases H have been implicated in DNA replication and recombination, and repair, their physiological roles are not completely understood. In vitro, the enzymes will also bind double-stranded (ds) DNA, ssDNA, ssRNA, and dsRNA, albeit with lower affinities than they bind to RNA:DNA hybrids.
The present inventors found a way to exploit the ability of proteins that recognize and bind to RNA:DNA hybrids in order to provide a basis for novel methods to quantify specific RNA sequences in a mixed or pure population of RNA molecules. The present inventors also contemplate the use of proteins that recognize and bind RNA:DNA hybrids in other novel applications, such as the capture of whole families of RNAs all containing the same or closely-related nucleotide sequences.
Furthermore, the present inventors found a way to make the RNA:DNA hybrid binding protein, RNase H, more useful for the methods suggested in the preceding paragraph, by overcoming or minimizing the following problems of the RNase H enzymes of the art.
A wild-type RNase H has RNA-degrading activity, which can pose a problem for applications to the recognition of RNA:DNA hybrids such as those contemplated by the present inventors. For example, RNA-degradation may degrade the RNA to be specifically detected in RNA:DNA hybrids. For this reason, the RNA:DNA hybrid recognizing antibody methods of the art typically use enzymes, such as reverse transcriptase, that is lacking in this exonuclease aspect of RNase H activity. Further, a wild-type RNase H binds other types of nucleic acid in addition to RNA:DNA hybrids. In the methods of the present invention, it is preferable that binding of RNA:DNA hybrids is enhanced over other kinds of nucleic acid binding, such as single stranded nucleic acid. Accordingly, there is room for improvement of the discrimination between RNA:DNA hybrids over other kinds of duplex nucleic acid.
Due to the ubiquity of the enzyme, RNase H, there are several sequences for RNase H known in the literature. There are several RNase H enzymes known in the art, and their amino acid sequences vary widely. U.S. Pat. No. 5,268,289 discloses a thermostable RNase H, as does U.S. Pat. No. 5,500,370. U.S. Pat. No. 6,376,661 discloses a human RNase H and compositions and uses thereof. U.S. Pat. No. 6,001,652 discloses a human type 2 RNase H. U.S. Pat. No. 6,071,734 discloses RNase H from HBV polymerase.
The protein sequence database, NCBI (National Center for Biological Information), lists several references for submitted protein sequences that are identical to the E. coli RNase H of SEQ ID NO:1, e.g. gi24111645 and gi24050418 (matching all 155 of the 192 residues listed); gi15799890, gi15829464, gi16128201, gi133163, gi17311, gi443433, gi443227, gi1942322, gi42062, gi42777, gi147680, and more (matching 155 of 155 residues listed); gi1942213 discloses an alteration of residue 134 from aspartic acid (D) to anything; gi1942211 discloses the mutation from D (aspartic acid) to A (alanine). An early nucleotide sequence in the public database for RNase H1 has a point error that results in one too few cysteine residues in the protein.
In counterpoint to the present invention, the art teaches several RNA detection methods that utilize reverse transcriptase lacking RNase H biochemical activity. Examples include those methods disclosed in U.S. Pat. Nos. 6,277,579 and 5,994,079 “Direct Detection of RNA mediated by Reverse Transcriptase lacking RNAse H Function.” U.S. Pat. Nos. 5,668,005 and 5,405,776 disclose genes for reverse transcriptase lacking RNase H activity.
In another area of the art, antisense nucleic acid methods, RNase H is utilized to cleave RNA. See Published U.S. Patent Application No. 20010044145, published Nov. 22, 2001, “Methods of using mammalian RNase H and compositions thereof,” which teaches a method of promoting inhibition of expression of a selected protein by an antisense oligonucleotide targeted to an RNA encoding the selected protein, wherein RNase H binds to an oligonucleotide-RNA duplex and cleaves the RNA strand to promote inhibition of protein expression.
In summary, there is a need for an RNase H that has less RNA-degrading (nucleolytic) activity. There is a need for an RNase H with enhanced binding to RNA:DNA hybrids. There is a great demand for improved discrimination between RNA:DNA hybrids and other forms of nucleic acid, such as ssDNA, ssRNA, dsDNA and dsRNA.